Herpes Simplex Virus (HSV) is a well-studied virus. Both distinguishable serotypes of Herpes Simplex Virus (HSV-1 and HSV-2) cause infection and disease ranging from relatively minor fever blisters on lips to severe genital infections, and generalized infections of newborns. HSV-1 and HSV-2 are 50% homologous at the DNA level, and polyclonal antibodies and MAbs to shared epitopes for one are cross-reactive to the other.
HSV-1 and HSV-2 have RR1 proteins (respectively designated ICP6 and ICP 10) that contain a unique amino terminal domain. The HSV-2 unique domain codes for a ser/thr-specific PK which has auto- and transphosphorylating activity and has a transmembrane (TM) domain. Sequences which code for the PK domain cause neoplastic transformation and are associated with cervical cancer (HSV-2 oncogene). The unique terminal domain of the HSV-1 RR1 protein (ICP6) also has PK activity but it is different from that of the HSV-2 oncoprotein, both structurally and functionally.
Original studies, using enzymatic assay conditions similar to those employed for ICP10 PK, concluded that ICP6 does not have PK activity, although the unique domain is retained (Chung et al., J. Virol. 63:3389-3398, 1989). This was not unexpected since the sequence of the unique PK domains showed only 38% homology (Nikas et al., Proteins: Structure, function and genetics 1:376-384, 1986). Further studies indicated that ICP6 has PK activity but only under different conditions. There are conflicting results about its ability to transphosphorylate other proteins (see Peng et al., Virology 216:184-196, 1996 for a review of the problem; particularly Table 1). The reason for the different PK activities of the ICP6 and ICP 10 proteins is likely to be that the ICP6 PK ATP binding sites are located distantly from the rest of the catalytic motifs (Cooper et al., J. Virol 69:4979-4985, 1995). ICP6 also does not have a functional TM domain and it does not localize to the cell surface (Conner et al., Virology 213:615, 1995). The PK activity of the native ICP6 is very weak even under ideal conditions, such that its K.sub.m is 10-fold higher than that of ICP10 PK (Peng et al., Virology 216:184, 1996; Lee and Aurelian, in preparation).
The transforming activity of ICP6 is located within a genome fragment that is distant from that at which the HSV2 oncogene is located. Transformation in this system is morphologic (focus forming ability).
It has previously been shown that DNA sequences which encode for the amino-terminal one-third of ICP 10 (amino acids 1-417) have oncogenic potential. Cells transfected with these DNA sequences evidence anchorage independent growth and cause tumors in animals. Transformation is seen in both rodent and human cells (Jariwalla et al., PNAS 77:2279-2283, 1980; Hayashi et al, PNAS 82:8493-8497, 1985; Smith et al., Virology 200:598-612, 1994; Hunter et al., Virology 210:345-360, 1995).
There are three functional domains within ICP10 amino acids 1-411: (i) an intracellular domain, at amino acids 106-411, which encompasses the PK catalytic domain with eight conserved catalytic motifs (amino acids 106-411), (ii) a TM, at amino acids 88-105, and (iii) an extracellular domain at amino acids 1-88 (Chung, et al., J. Virol. 63:3389-3395, 1989; Virology 179:168-178, 1990). The minimal size required for PK activity is amino acids 1-283 (pp29.sup.la1) (Luo et aL, J. Biol. Chem. 266: 20976-20983, 1991). However, the PK activity of pp29.sup.la1 has some properties different from the authentic ICP10 PK, presumably because it lacks part of the PK catalytic domain VI (Luo et al., J. Biol. Chem. 266: 20976-20983, 1991). The TM domain is also required (but insufficient) for PK activity (Luo and Aurelian, J. Biol. Chem. 267:9645-9653, 1992). Therefore, it can be concluded that the PK activity is localized within amino acids 88-411 with an essential core at amino acids 88-283.
The unique HpaI site within the ICP10 coding region represents the 3' end of the transforming region (Jariwalla et al., Proc. Nat. Acad. Sci. 77:2279-2283, 1980) and cuts the gene after the codon for amino acid residue 417. It is not known whether pp29.sup.la1 has transforming activity. However, PK activity is required for neoplastic potential. PK negative mutants do not transform cells. This includes a mutant deleted in the TM domain and site directed mutants in the ATP binding sites (Lys.sup.176 and/or Lys.sup.259) or the ion-binding site (Glu.sup.209) (Smith et al., Virology 200:598-612, 1994; Aurelian, L. Frontiers in Biology, in press). Because a PK mutant deleted only in the TM domain does not have transforming activity (Smith et al., Virology 200:598-612, 1994), DNA sequences that code for ICP10 amino acids 106-411, but lack PK activity, are not intrinsically neoplastic. This demonstrates that: (i) the HSV-2 oncoprotein is located within ICP10 amino acids 1-411, and (ii) neoplastic potential requires a functional PK activity.
The function of ICP10 PK in virus growth/pathogenesis is unknown.
The HSV-2 ICP10 protein has intrinsic PK activity. This was shown by demonstrating that ICP10 PK activity is lost through site-directed mutagenesis. The oncogene also has SH3-binding sites at positions 140, 149 and 396, which are required for interaction with signaling proteins. This interaction is required for transforming activity. Site directed mutagenesis was used to identify amino acids required for kinase activity and interaction with signaling proteins. Mutation of Lys.sup.176 or Lys.sup.259 reduced PK activity (5-8 fold) and binding of the .sup.14 C-labeled ATP analog p-fluorosulfonylbenzoyl 5'-adenosine (FSBA), but did not abrogate them. Enzymatic activity and FSBA binding were abrogated by mutation of both Lys residues, suggesting that either one can bind ATP. Mutation of Glu209 (PK catalytic motif III) virtually abrogated kinase activity in the presence of Mg.sup.2+ or Mn.sup.2+ ions, suggesting that Glu.sup.209 functions in ion-dependent PK activity.
ICP10PK functions as a growth factor receptor involved in signaling and it binds the adaptor protein Grb.sub.2 in vitro. The SH3-binding sites within the ICP10 PK domain (at positions 140, 149 and 396) are required for interaction with signaling proteins and, thereby transformation (Nelson et al., J. Biol. Chem. 271:17021-17027, 1996). Mutation of the ICP10 proline-rich motifs at position 396 and 149 reduced Grb.sub.2 binding 20- and 2-fold respectively. Binding was abrogated by mutation of both motifs. Grb.sub.2 binding to wild type ICP10 was competed by a peptide for the Grb.sub.2 C-terminal SH3 motif indicating that it involves the Grb.sub.2 C-terminal SH3 (Nelson et al., J. Biol. Chem. 271:17021-17027, 1996).
The ICP10PK catalytic domain also contains amino acids at position 106-178 that are responsible for binding a down-regulator of PK activity (ras-GAP). Deletion of amino acids 106-178 reduces, but does not abrogate, PK activity (Luo and Aurelian, J. Biol. Chem. 267:9645-9653, 1992). However, it abrogates ras-GAP binding, thereby increasing transforming potential (Nelson et al., manuscript in preparation).
The construction of the ICP10 PK virus is described by Peng et al. (Virology 216, 184-196, 1996). Briefly, the wild type sequences in a plasmid (TP101) that contains the HSV-2 BamHI E and T fragments were replaced with the 1.8kb SalI/BglII fragment from pJHL9. pJHL9 is a plasmid containing the ICP10 mutant deleted in the PK catalytic domain (Luo and Aurelian, J. Biol. Chem. 267:9645-9653, 1992). The resulting plasmid, TP9, contains sequences which code for ICP10 deleted in the PK catalytic domain flanked by 4 and 2.8 kb of HSV-2 DNA sequences at the 5' and 3' ends, respectively. The 10 kb HindIII/EcoRI fragment from TP9 was introduced by marker transfer into a virus (ICP10.increment.RR) in which the RR domain of ICP10 had been replaced with the LacZ gene. The resulting recombinant virus, designated ICP10.increment.PK, was obtained by selecting white plaques on a background of blue plaques after staining with X-gal. A few white plaques were picked and purified. Two were grown in Vero cells with 10% serum (exponentially growing) into individual stocks respectively designated RF and CS.
There are several known HSV vaccines in the prior art. U.S. Pat. Nos. 4,347,127; 4,452,734; 5,219,567; and 5,171,568 each teach subunit vaccines which provide some protection against HSV-2 infection. These vaccines are inferior to one in which a live, attenuated virus is used. The immunity induced by a subunit vaccine is restricted to the particular protein represented by the subunit, which may not have sufficient protective potential. Additionally it is non-replicating and there is, therefore, no amplification of the protein which would further reduce immunogenicity. These problems occur in any subunit vaccine regardless of whether the method of preparation is via a recombinant protein or the purification of an antigen from the virus.
A cross recombinant vaccine, such as disclosed in U.S. Pat. No. 4,554,159, does not suffer from the problems of the subunit vaccines, but contains the oncogene present in HSV-2. Unless care is taken to define and delete the oncogene, the cross recombinant vaccine would induce cancer in the vaccinee.
The cross recombinant of '159 is temperature sensitive. Avirulence may be obtained by selecting temperature resistance, but the temperature of the mouse is 39.degree. C. while that of humans is 37.degree. C. This temperature sensitivity could well render such a cross problematic in a vaccine. A superior method of selection of avirulence is by the removal of genes coding for virulence without respect to the temperature at which the virus replicates. Also, the use of prototypical crosses would preclude the use of mutants with deleted or inserted genes.
Due to the many type-common epitopes on HSV-1 and HSV-2, the antibodies in human serum are cross-reactive (Aurelian, et al., J. Natl. Cancer Inst. 45:455-464, 1970.) It has also been previously shown that cell-mediated immunity cross-reacts (Jacobs et al., J. Immunol. 116:1520-1525, 1976).
A live vaccine is superior to a dead vaccine because the live vaccine induces herd immunity and it also induces different types of immunity, such as mucosal, cell mediated and humoral immunity. A higher level of immunity is normally obtained because the virus titers are increased through replication within the vaccinee. Finally a live vaccine is of longer duration, thus obviating boosters and lowering initial dosage. However, an absolute necessity for a live herpes vaccine is the removal of the gene responsible for causing transformation, as in the present invention.
Known vaccines are not virus type-specific. All known vaccines for HSV-1 or HSV-2 are cross-reactive and provide immunity to the other virus type. Most developed vaccines (viz. those in neurovirulence genes) are in HSV-1. However, HSV-1 is not as desirable a vaccine candidate against herpes, because the major clinical problem is the sexually transmitted HSV-2, which is also associated with cancer induction. Recent studies indicate that the age-adjusted prevalence of HSV-2 in the US is now 20.8%, an increase of approximately 30% over the past 13 years (Fleming et al., New Engl. J. Med. 337:1105-1111, 1997). The increasing rate of HSV-2 acquisition among young adults increases the likelihood that infants will be exposed to HSV-2 at delivery, resulting in an infection that, despite antiviral therapy, is still life-threatening (Whitley, et al., New Engl. J. Med. 327:782-799, 1992 [Erratum, N.Engl. J. Med. 328:671, 1993]). A new concern about HSV-2 infection is that it may facilitate the spread of HIV and increase the severity of the disease (Aurelian, L. Editor. Herpes viruses, the Immune System and AIDS. Kluwer Academic Publishers, Boston, Mass. 1990). Because HSV-1 has only a 50% homology to HSV-2, this may lower the response rate against the heterologous strain in the vaccinated population.
Another absolute requirement for a live vaccine is the absence of lesions upon immunization. A desirable trait in the live vaccine would be its ability to cause a reduction in the frequency of recurrent lesions in a person already infected. There is a substantial population already infected with HSV who may have intercourse with uninfected individuals who would benefit from such a vaccine.
The present invention solves all the problems recited above providing a whole live attenuated HSV-2 in which the HSV-2 has a deletion of the oncogene, and is formulated in a vaccine composition. The present invention provides a method of immunizing a subject against HSV-1 or HSV-2 with said vaccine composition, providing a superior method of conferring immunity upon the subject.